A Two-Photon-Active Zr-Based Metal–Organic Framework-Based Orthogonal Nanoprobe for Recognition of Cellular Senescence

A luminescent nanoprobe capable of orthogonal sensing of two independent events is highly significant for unbiased disease-related detection such as the detection of senescent cells. Moreover, it is invaluable that the nanoprobe possesses a two-photon excitable characteristic that is highly suitable for imaging living cells and tissues. Herein, we present a two-photon-excitable multiluminescent orthogonal-sensing nanoprobe (OS nanoprobe) capable of detecting both pH elevation and β-galactosidase (β-gal) overexpression in senescent cells. In the design, Zr-based dual-emissive metal-organic frameworks prepared from two mixed amino linkers, referred to as NH₂-MU, were used as the component for the ratiometric sensing of pH; additionally, fluorogenic resorufin-β-d-galactopyranoside, linked to the NH₂-MU framework, enables β-gal detection. In the OS nanoprobe, the signals for pH and β-gal sensing remain independent while maintaining high colocalization. The two-photon excitable organic linkers of NH₂-MU impart the OS nanoprobe with a bioimaging capability, allowing for the differentiation of senescent human foreskin fibroblast (HFF) cells from younger HFF cells or LacZ positive cells with the 800 nm laser excitation. This study marks the first instance of achieving the multiplexed orthogonal fluorescent sensing of cellular senescence using a two-photon excitation strategy, suggesting the potential of using versatile metal-organic framework (MOFs)-based fluorophores to realize the orthogonal multiplexing of disease-related biomarkers through multiphoton excitation.